Phosphoenolpyruvate carboxylase (PEPC) is essential for the glycolytic pathway and parasite proliferation in Babesia gibsoni

Abstract Apicomplexan parasites predominantly generate ATP and lactic acid through glycolysis and anaerobic glucose metabolism, incorporating CO 2 into glycolysis via a stage-dependent phosphoenolpyruvate carboxylase (PEPC) mechanism. Although the role of PEPC in plant and bacterial carbon fixation is well documented, its function within Babesia remains largely unexplored. This study employs reverse genetics to probe the biological role of PEPC in Babesia gibsoni , noting its conservation across similar protozoa, suggesting a pivotal and conserved biological function. Western blotting and immunofluorescence (IFA) experiments using the BgPEPC-3 × Flag strain revealed that the BgPEPC protein has a molecular weight of 105 kDa and localizes predominantly to the cytoplasm. Attempts to knock out the PEPC gene in BgPEPC-3 × Flag strains failed under standard media conditions, succeeded only with the addition of 5 mM malate, an upstream metabolite of oxaloacetic acid (OAA). In addition to malate, the downstream metabolite of OAA can also partially compensate for the phenotypic defects caused by PEPC deficiency. This intervention alleviated severe growth deficits, underscoring the critical role of aspartate in the parasite lifecycle. Moreover, metabolic inhibitors such as L-cycloserine and triazamidine, which target aspartate aminotransferase and mitochondrial functions, respectively, demonstrated increased efficacy against BgPEPC knockout strains. The lack of a compensatory response to malic acid supplementation underscores the integral role of BgPEPC in intermediary carbon metabolism and its necessity in providing aspartate as a precursor to pyrimidine synthesis. Collectively, these findings suggest that PEPC could be a potential target for future drug development against B. gibsoni infections. Graphical Abstract